Identification of AP Gene Family and Its Response Analysis to Abiotic Stress in Setaria italica

doi:10.13560/j.cnki.biotech.bull.1985.2023-0588

Abstract

Abstract:

Aspartic protease（AP）is a pivotal hydrolase enzyme that assumes a critical role in plant growth, development, resilience against both biological and abiotic stressors. Foxtail millet（Setaria itlica）is the model crop for stress resistance study of C₄ gramineous plants, but there are few studies on the gene function of AP（SiAPs）in foxtail millet. In order to further explore the functional role of the APs in foxtail millet, the members of the SiAPs were indentified based on whole-genome screening of SiAPs conserved Pfam sequences, and bioinformatics method was used to analyze their physical and chemical properties, subcellular localization, gene structure, conserved domain, phylogenetic development, promoter cis-acting elements and collinearity by bs. Meanwhile, fluorescence quantitative PCR technique was used to study its expression mode under abiotic stress. The results showed that there were 58 AP gene family members in foxtail millet genome. Phylogenetic tree analysis demonstrated that the gene family could be divided into 5 subfamilies, among which Group B encoded atypical AP, while other subfamilies encoded nucellin-like AP. The analysis of gene structure and conserved motifs revealed that members of the same subfamily of APs in foxtail millet were highly conserved. Collinearity analysis showed that there were a large number of homologous gene pairs between SiAPs and rice（Oryza sativa）and maize（Zea mays）AP genes. The study of promoter cis-acting elements indicated that most members of SiAPs contained cis-acting elements related to abiotic stress and biohormone response, such as cis-acting elements in response to drought and low-temperature stress, and salicylic acid response elements. Further quantitative PCR results uncovered that SiAPs were differentially expressed in the root, stem, leaf and panicle of foxtail millet. The expressions of SiAP3, SiAP9 and SiAP48 genes significantly increased under low temperature stress. Under drought stress and SA treatments, the change trend of some gene expression was basically the same. SiAPs play an important role in regulating foxtail millet response to abiotic stress. The results of this study may provide reference for stress resistance analysis of SiAPs.

Key words: foxtail millet（Setaria itlica）, aspartate protease, salicylic acid response, drought response, low-temperature stress, bioinformatics

XING Yuan, SONG Jian, LI Jun-yi, ZHENG Ting-ting, LIU Si-chen, QIAO Zhi-jun. Identification of AP Gene Family and Its Response Analysis to Abiotic Stress in Setaria italica[J]. Biotechnology Bulletin, 2023, 39(11): 238-251.

Figures/Tables 12

Table 1 Sequences of primers

基因名称Gene name	序列Sequence（5'-3'）
25S-F	AGGCAACAGAAACTCCATACG
25S-R	ATGGCATAGCATTCATCACG
SiAP2-F	AACATACCCACCTCGGTCTTC
SiAP2-R	CGCCTTGTACTTCTTCATCCC
SiAP3-F	GATACCTGCTACGACTTCACCG
SiAP3-R	GCCGACATCATAGAGCACCTC
SiAP4-F	ATGTCAGAGGGCGGCTACG
SiAP4-R	TGGTGAGGCAGTACGAGAAGG
SiAP9-F	TCCAGTCCACGCCGCTTATC
SiAP9-R	CGAAGATGATGCCGCCACTC
SiAP32-F	GGTGGTGGGCGGCAACTC
SiAP32-R	CCAGCAGGCGGTTCTCCATC
SiAP36-F	GCTGTCGTCGGCGTTCAAG
SiAP36-R	GTCGGTATCGTGATGCTTCTCTG
SiAP48-F	CATCAGCCAGAGGTGCCAGAG
SiAP48-R	CGTGTTGGTGTAGTCGTCGTATTG

Fig. 1 Gene structure and conservative motif analysis of members of SiAPs in Setaria italic A: Cluster analysis of AP gene family members in foxtail millet（Seitaria italic）. B: Conservative domain of AP gene family members in foxtail millet. C: Gene structure map of SiAPs members in foxtail millet. D: Logo of conserved motif of SiAPs in foxtail millet

Fig. 2 Unrooted phylogenetic tree of AP gene family in Seitaria italic（Si）, Oryza sativa（Os）and Arabi-dopsis thaliana（At）

Fig. 3 Distribution of AP gene family members on chromosomes in S. italic 1-9 represents chromosome 1-9 in S. italic, respectively. The interior of the chromosome is filled with gene density, with colors ranging from blue to red indicating a gradual increase in gene density

Fig. 4 Collinearity analysis of AP gene family in S. italic 1-9 represents chromosome 1-9 of S. itlica. The heat map represents gene density, with increasing gene density from blue to red

Fig. 5 Collinearity analysis of SiAPs gene family members and AP genes in A. thaliana, O. sativa and Z. mays A: The synteny analysis of AP genes in S. italica and Z. mays; B: the synteny analysis of AP genes in S. italica and O. sativa; C: the synteny analysis of AP genes in S. italica and A. thaliana

Fig. 6 Statistics of cis-acting elements of AP gene family in S. italic

Fig. 7 Cis-acting elements analysis in the promotors of AP gene family in S. italica

Fig. 8 Expression analysis of AP gene family members in the different tissues of S. italica

Fig. 9 Expression analysis of AP gene family members in S. italica induced by 0.25 mmol/L salicylic acid Error bars are standard errors. The different letters in the figure indicate significant differences（P < 0.05）. The same below

Fig. 10 Expression analysis of AP gene family members in response to drought stress

Fig. 11 Expression analysis of AP gene family members in response to low temperature stress

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